Sensor

ABSTRACT

A sensor for detecting and/or quantifying the amount of analyte in a sample, the sensor including:
         a sensing region; and   a barrier layer including a reactive oxygen species (ROS)-quenching, analyte-permeable membrane having an ROS-quenching agent adsorbed to the membrane;   wherein the sensor is adapted so that the sample enters the sensing region of the sensor through the barrier layer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application SerialNo. PCT/GB2013/050407, having an international filing date of Feb. 20,2013, which claims the benefit of U.S. Provisional Application Ser. No.61/611,239, filed Mar. 15, 2012.

FIELD

This disclosure relates to sensors, membranes for use in the sensors,methods for making such sensors and membranes and methods for detectingor determining the quantity of glucose in a sample.

BACKGROUND

Molecular receptors such as boronates can be used in sensors for thedetection and/or measurement of analyte in biological fluids. Forexample, a sensor may comprise a glucose receptor (the boronic acid) anda fluorophore that acts as the transmitter of the signal. Theseindicator chemistries can readily be immobilised onto an optical fibreof appropriate diameter, which can then be placed into body fluids ortissue to measure analytes such as glucose.

SUMMARY

Under oxidative stress, it has been found that levels of reactive oxygenspecies (ROS) such as hydrogen peroxide (H₂O₂) can rise. Oxidativestress can arise as a result of an ischemic event or sepsis (e.g. as aresult of multi-organ failure) and is also implicated in many diseases(e.g. atherosclerosis, Parkinson's disease, heart failure, myocardialinfarction, Alzheimer's disease, schizophrenia, bipolar disorder,fragile X syndrome and chronic fatigue syndrome), thereby raising levelsof ROS in the body fluids or tissue of subjects who may require theirglucose levels to be monitored, for example in an intensive careenvironment. ROS in the blood could therefore interfere with sensorindicating chemistry.

It has been found that including a ROS-quenching agent in the sensorcould eliminate or ameliorate the interferent effect on sensor chemistryof ROS in the blood. Accordingly, a sensor for detecting and/orquantifying the amount of analyte in a sample can include:

-   -   a sensing region; and    -   a barrier layer including a reactive oxygen species        (ROS)-quenching, analyte-permeable membrane having an        ROS-quenching agent adsorbed thereto;        wherein the sensor is adapted so that the sample enters the        sensing region of the sensor through said barrier layer.

The presence of ROS-quenching agents in sensors can oxidise (orotherwise deplete) analyte, which can adversely affect the sensoroperation. For example, glucose may be oxidised to gluconic acid. Thesensor can thus include a means to address the problem of analyteoxidation in sensors including a ROS-quenching agent. In someembodiments, therefore, the membrane of the sensor can selectivelyquench ROS over analyte.

In some embodiments, the sensor includes a reactive oxygen species(ROS)-quenching, analyte-permeable membrane, suitable for use in asensor for detecting and/or quantifying the amount of analyte in asample, the membrane having an ROS-quenching activity sufficient toquench a solution of H₂O₂ having a concentration of 100 ppm or less.

A process for producing a ROS-quenching analyte-permeable membranesuitable for use in a sensor for detecting and/or quantifying the amountof analyte in a sample is also described. The process includes:

-   -   (iii) contacting a barrier layer with a ROS-quenching precursor        and a reducing agent;    -   (iv) reducing the ROS-quenching precursor to form a        ROS-quenching agent on or in the barrier layer; and optionally        repeating steps (iii) and (iv).

A membrane obtainable or obtained by this process is also described.

In one embodiment the process further comprises, before step (iii):

-   -   (i) contacting a barrier layer with a ROS-quenching precursor        and a preliminary reducing agent;    -   (ii) partially reducing the ROS-quenching precursor to form a        ROS-quenching agent on or in the barrier layer; and        optionally repeating steps (i) to (iv). Thus, in this        embodiment, the process includes:    -   (i) contacting a barrier layer with a ROS-quenching precursor        and a first reducing agent;    -   (ii) partially reducing the ROS-quenching precursor to form a        ROS-quenching agent on or in the barrier layer;    -   (iii) contacting the barrier layer with a second reducing agent;    -   (iv) fully reducing the remaining ROS-quenching precursor to        form a ROS-quenching agent on or in the barrier layer; and        optionally repeating steps (i) to (iv). A membrane obtainable or        obtained by this process is also described.

The process described herein is particularly beneficial in that itachieves deposition of the ROS-quenching agent within the pores of amembrane, rather than merely on the surface of the membrane. Suchdeposition within the pores can be achieved even where the membrane hasa high aspect ratio, for example an aspect ratio of at least 100,typically at least 150 or 200. Deposition of the ROS-quenching agentwithin the pores leads to an improved ability of the membrane to quenchROS, since fluid passing through the membrane will be in contact with anROS-quenching agent throughout the time it is moving through the pores.

A method of detecting and/or quantifying the amount of analyte in asample is also described. The method can include inserting into thesample a sensor as described herein, providing incident light to thesensing region of the sensor, and detecting the emission pattern of thefluorophore.

Further preferred features and embodiments are described in theaccompanying description and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 and 1 a depict a sensor incorporating an optical fibre and amonitor for such a sensor.

FIGS. 2, 3 and 3 a depict various embodiments of a sensing region of asensor.

FIG. 4 depicts a schematic version of an apparatus which may be used todetermine ROS quenching ability and/or analyte depletion for a membrane.

FIGS. 5A to 5D depicts SEM images for the membrane of Example 2.

FIG. 6 depicts the results of in vitro testing of a sensor and acorresponding sensor without a platinised membrane.

FIG. 7 depicts the results of Example 4.

FIG. 8 depicts a schematic view of the process for producing aROS-quenching barrier layer

FIG. 9 depicts the results of in vivo testing of a sensor (FIG. 9 b) anda corresponding sensor without a platinised membrane (FIG. 9 a).

FIG. 10 depicts the results of Example 9. Results for the threeplatinised sensors are shown as IB070-001, IB070-002, and IB070-003.Results for the unplatinised control are shown as IB066-011.

DETAILED DESCRIPTION

As used herein an ROS-quenching, analyte-permeable barrier layer is amaterial which allows the passage of analyte through the layer but whichrestricts or preferably prevents ROS from passing through the layer,typically by catalysing its decomposition into chemical species which donot interfere with sensor chemistry (e.g. boronic acid/fluorophorechemistry).

The ROS-quenching, analyte-permeable barrier layer is envisaged for usewith any sensor. In some embodiments, the sensor is a glucose sensor,preferably an optical glucose sensor using boronic acid/fluorophoreglucose sensing chemistry. The sensor can also be an electrochemical orpH sensor.

The ROS-quenching, analyte-permeable barrier layer may be used with anyoptical sensor having an optical waveguide for directing incident lightonto the sensing region of the sensor. For example, the optical sensorcan be a fibre optic sensor. Glucose sensing can be carried out inbodily fluids such as interstitial tissue or blood, although sensing ofany aqueous solution may be carried out using the sensors describedherein. The particular embodiments described herein are envisaged foruse as invasive sensors for insertion into a blood vessel. However, theuse of the ROS-quenching, analyte-permeable barrier layer is not limitedto such invasive sensors. Non-invasive sensors for in vitro use,implantable sensors, and subcutaneous sensors can also include aROS-quenching, analyte-permeable barrier layer.

As used herein, the words “include” and “contain”, and variationsthereof such as “includes”, “contains”, “including” and “containing”,are used in an inclusive sense, i.e. to specify the presence of thestated features but not to preclude the presence or addition of furtherfeatures.

The Barrier Layer

The barrier layer comprises an ROS-quenching, analyte-permeable membranehaving an ROS-quenching agent adsorbed thereto. The ROS-quenching agentmay be adsorbed to the surface of the membrane (e.g., the membrane iscoated with the ROS-quenching agent), or may be adsorbed to the pores ofthe membrane (e.g., the membrane may be impregnated with theROS-quenching agent).

Suitable analyte-permeable membranes which can be coated or impregnatedwith an ROS-quenching agent and used as the barrier layer in the sensorinclude semi-permeable membranes such as dialysis membranes andmicroporous hollow fibre membranes.

In some embodiments, the barrier layer is hydrophilic.

Dialysis membranes are semi-permeable membranes that separate moleculesby virtue of their size, shape, hydration and polarity. Dialysismembranes are usually in the form of hollow fibres and are available inmaterials such as polyarylethersulphone, polyamide, polycarbonate,polyacrylonitrile, polysulphone, polyethersulphone,polyvinylidenefluoride and cellulosic materials or mixtures ormodifications thereof.

Microporous hollow fibre membranes are known in the art and includepolypropylene hollow fibre membranes. For example, a polypropylenehollow fibre membrane having a pore size of from 0.1 to 0.2 μm, with aporous area of approximately 40% of the surface, a minimum internaldiameter of the fibre of 416 μm and a maximum outer diameter of thefibre of 510 μm can be coated or impregnated with an ROS-quenching agentand used as the barrier layer.

In one aspect of this embodiment, a polymer, e.g. a hydrophilic and/ornegatively charged polymer, is present within the pores of the membrane.This can be achieved via in situ polymerisation, within the pores of themembrane, of a monomer mixture, for example including one or morehydrophilic monomers and/or one or more negatively charged monomers.Suitable in situ polymerisation techniques are described ininternational patent application number PCT/GB2011/000209, the contentof which is incorporated herein by reference.

In other embodiments, the membrane does not have a hydrophilic polymerpresent within its pores.

In some embodiments, the membrane used to form the barrier layer in thesensor is a dialysis membrane or a polypropylene hollow fibre membrane.

A semi-permeable membrane used in the barrier layer typically has a poresize of from 1 nm to 1 micron, e.g. from 1 to 20 nm or from 0.1 to 0.5micron. Typically the hollow fibre polypropylene membrane used to formthe barrier layer of the present sensor will have pore dimensions offrom 0.1 micron to 0.5 micron, e.g. 0.1 to 0.3 micron such as about 0.2micron. The dialysis membrane used to form the barrier layer of thepresent sensor will typically have pore dimensions of 100 nanometers orless. Preferred pore sizes are 1 to 20 nm, preferably 1 to 10 nm, forexample about 6 nm.

In some embodiments, the membrane has an aspect ratio of at least 100,preferably at least 150, for example at least 200 or at least 250. Asused herein, the aspect ratio is the ratio of the length of each pore(i.e. the thickness of the membrane) divided by the pore diameter.Advantageously, the ROS-quenching agent is distributed along the lengthof the pore (i.e. not only at the opening of the pore or a part of thepore adjacent the opening of the pore). The techniques for producing theROS-quenching membrane described are particularly effective atfacilitating distribution of the ROS-quenching agent along the length ofthe pore. The thickness of the membrane is typically at least about 20micron, for example at least about 30 micron. The thickness may be up toabout 50 micron, for example up to about 40 micron. Typically in thisembodiment the membrane is a hollow fibre polypropylene membrane.

Semi-permeable membranes are available with different pore sizesrelating to the molecular weight cut-off (MWCO) of the membrane. Themolecular weight cut-off indicates the maximum molecular weight ofmolecule which can pass through the pores of the membrane. The semipermeable membrane used in the present sensor has a MWCO such that theanalyte can pass through. The semi permeable membrane used in thepresent sensor is typically a low MWCO material that does not allowmaterials of molecular weight 6,000 or higher (e.g. to proteins) to passthrough, but does allow glucose (MW180) to pass. Preferred membraneshave a MWCO of at least 1,000 and preferably no more than 5,000. Forexample, the MWCO may be at least 1,500 or at least 2,000, for exampleno more than 4,000.

The effective pore size and MWCO of the final barrier layer used in thesensor (herein “effective pore size”, “effective MWCO”) may be lowerthan those described above as a result of in situ polymerisation, orloading of the ROS quenching agent. The effect of in situ polymerisationon pore size is described in PCT/GB2011/000209 (referenced above), thecontent of which is incorporated herein by reference. Preferredeffective pore sizes for the final membrane are at least 1 nm, e.g. atleast 2 nm or at least 4 nm and no more than 20 nm, e.g. no more than 10nm. Preferred effective MWCO are at least 1,500 or at least 2,000 and nomore than 6,000 e.g. no more than 5,000 preferably no more than 4,000.

Measurement of the pore size, or effective pore size, can be carried outby any method known to the skilled person. Typically, the pore size isgiven as the median pore size for any particular membrane. MWCO can bedetermined by the diffusion of monodisperse materials of known molecularweights with a fluorescent molecule attached. Materials of graduallyincreasing molecular weight are passed through the membrane and thediffusion breakthrough can be determined using a fluorimeter as adetector. Examples of suitable monodisperse materials arefluorescein-labelled dextrans available from Sigma-Aldrich in a varietyof molecular weights. The effective pore size or effective MWCO may bemeasured by preparing the final membrane and measuring the pore size orMWCO in the usual way.

In order to provide an acceptable response time (for instance for anintravascular sensor which continuously measures glucose), the barrierlayer should preferably be selected so as to provide a 90% response timeof a sensor which is no more than three minutes, preferably no more thantwo-and-a-half minutes.

The 90% response time is determined as the time taken from addition of aknown amount of analyte to a sample, to the sensor response reaching 90%of the analyte concentration. This can be measured by contacting thesensor with a zero analyte aqueous solution, adding a known amount ofanalyte at time t₀ and monitoring the sensor response over time. Thesensor response increases over time and the time after t₀ at which thesensor reading corresponds to 90% of the added analyte concentration istaken as the 90% response time. In this technique, the analyte is addedin such a manner that the change in concentration of the aqueoussolution is substantially instantaneous, so there is no time delay dueto, for example, dissolution of the analyte. Thus, analyte is typicallyadded in liquid or concentrated solution form, with stirring.

ROS Quenching Agent

The ROS-quenching agent used in the barrier layer can be any substancecapable of catalysing the decomposition of reactive oxygen species suchas H₂O₂.

The decomposition of H₂O₂ into chemical species which do not interferewith sensor chemistry (e.g. boronic acid/fluorophore chemistry) canoccur by disproportionation to water and oxygen gas:2H₂O₂→2H₂O+O₂

Suitable substances capable of catalysing the decomposition of ROS suchas H₂O₂ include transition metals, transition metal compounds andenzymes.

Typically, the transition metal used as the ROS-quenching agent is ametal of Group 10 or 11 of the Periodic Table, e.g. nickel, palladium,platinum, copper, silver or gold. Preferably, the transition metal usedas the ROS-quenching agent is palladium, platinum, gold or silver. Morepreferably, the transition metal used as the ROS-quenching agent isplatinum. An alloy of two or more metals, such as an alloy of atransition metal of Group 10 or 11 with another metal, or an alloy oftwo or more

Group 10 or 11 transition metals, may also be used. Alloys of gold andsilver are particularly envisaged.

Typically, the transition metal compound used as the ROS-quenching agentis a compound of a metal of Group 7 of the Periodic Table, e.g. a Group7 oxide, for instance manganese dioxide.

Typically, the enzyme used as the ROS-quenching agent is catalase orsuperoxide dismutase, preferably catalase.

In a preferred embodiment, the ROS-quenching agent is a metal of Group10 or 11 of the Periodic Table or an alloy containing such a metal. In aparticularly preferred embodiment, the ROS-quenching agent is a metal ofGroup 10 or an alloy containing such a metal, more preferably platinumor a gold/silver alloy, most preferably platinum. These metals areparticularly useful as the ROS-quenching agent as they have an extremelylong lifetime in a device and degradation is not of concern. The metalscan also be simply adsorbed to the membrane (either to the surface orpreferably within the pores) Immobilisation, for example by covalentattachment of the quenching agent is not required.

When the ROS-quenching agent is a transition metal or a transition metalcompound, it is typically present in the membrane in the form ofnanoparticles, i.e. particles with a nanoscale average particle size,typically 1-100 nm, for example at least 5 nm, 10 nm or at least 20 nm,and for example up to 90 nm, 80 nm or 70 nm. Nanoparticulate materialsare advantageous since they can be provided within the pores of themembrane rather than solely on the surface. Their small size alsofacilitates an even distribution of the particles through the membranepores, leading to improved efficiency in quenching ROS.

In the case of nanoparticles, the particles may have any appropriateform or crystal structure. For example, platinum nanoparticles may be inthe form of tetrahedron, cube, octahedron, truncated cube,cuboctahedron, truncated octahedron, triangular plate, bipyramid,tripod, decahedron, rod or wire or icosahedron. Platinum nanoparticlesmay also be produced as spherical particles, hollow structures anddendrites. The various structures and techniques for their preparationare set out in Zhenmeng Peng, Hong Yang, Nano Today, 2009, 4, 143-164and the references cited therein. Brief details of the preparation ofthese different forms are set out in Table 1 below:

TABLE 1 Pt Nanoparticles Crystal Structures. Precursor Reductant^(a)Surfactant^(b) Additive^(c) Condition^(d) Shape^(e) K₂PtCl₄ H₂ Na[PA] pHRT, 12 h C, T K₂PtCl₄ H₂ Acrylic acid pH RT, 12 h C, T K₂PtCl₄ H₂ PNIPALCST C K₂PtCl₄ H₂ PNEA LCST Tri K2PtCl4 H₂ PVP, PNIPA, RT Tri, Sq,Na[PA] Hex K₂PtCl₆ H₂ Na[PA] RT C, TO K₂PtCl₆ H₂ PVP 25-45° C. TH₂PtCl₆, H₂ PVP RT, T K₂PtCl₄ overnight K₂PtCl₄ H₂ Na₃[Cit] NaOH RT C,T, Hex H₂PtCl₆ H₂ PVP RT, Tri, SF overnight K₂PtCl₆ H₂ PVP RT TetNa₂PtCl₄ PVP PVP 80° C. Tri, SP K₂PtCl₆ Na₃[Cit] Na[PA] Reflux, 3.5 h SPK₂PtCl₆ NaBH₄, H₂, TTAB 50° C. C, CO, AA PP H₂PtCl₆ NaBH₄ CTAB AgNO₃ RTC K₂PtCl₆ NaBH₄ CTAB HCl RT, 12 h DD H₂PtCl₆ EtOH PNIPA Reflux SPH₂PtCl₆ NaBH₄, H₂ Pluronic L64 RT SP H₂PtCl₆ NaBH₄ MSA SP K₂[Pt(C₂O₄)₂]H₂ K₂C₂O₄, CaCl₂ RT or 55° C. C, Hex K₂PtCl₄ K₂PtCl₆ K₂PtCl₄ Cu foilCu²⁺ C Na₂PtCl₆ Vitamin B2 Vitamin B2 RT SP H₂PtCl₆ Hydrazine AOTIsooctane RT SP K₂PtCl₄ γ-ray CTAB Hexanol RT NR H₂PtCl₆ Hydrazine Berol050 Isooctane RT SP K₂PtCl₄ UV, AA SDS, Brij-35, SnOEP, SP DSPC cholK₂PtCl₄ AgNR C/S H2PtCl₆ CoNP 95° C. Hol H2PtCl₆ H₂ Et-HMM 200° C. for 4h Nec H₂PtCl₆ ED RT SP K₂PtCl₄ ED AA H₂SO₄ RT THH K₂PtCl₆ ED H₂SO₄ RT NHNa₂PtCl₆ ED HCl RT NW K₂PtCl₆ ED H₃BO₃ RT NT H₂PtCl₆ γ-ray MeOH RT SPH₂PtCl₆ UV MeOH RT NW PtCl₄ Microwave α-Glucose SP Table 1. ^(a)PVP =poly(N-vinyl-2-pyrrolidone); Na₃[Cit] = sodium citrate; AA = ascorbicacid; EtOH = ethanol; NR = nanorod; NP = nanoparticle; ED =electrodeposition. ^(b)Na[PA] = sodium polyacrylate; PNIPA =poly(N-isopropylacrylamide); PNEA = poly(N-ethylacrylamide); TTAB =tetradecyltrimethylammonium bromide; CTAB = hexadecyltrimethylammoniumbromide; Pluronic L64 = EO₁₃PO₃₀EO₁₃ triblock copolymer; MSA =mercaptosuccinic acid; AOT = sodium bis(2-ethylhexyl)sulfosuccinate; SDS= sodium dodecylsulfate; DSPC =1,2-distearoyl-snglycero-3-phosphocholine. ^(c)MeOH = methanol; SnOEP =Sn(IV) octaethylporphyrin; chol = cholesterol. ^(d)LCST = lower criticalsolution temperature; RT = room temperature. ^(e)C = cube; T =tetrahedron; O = octahedron; THH = tetrahexahedron; CO = cuboctahedron;TO = truncated octahedron; SP = spherical particle; Tri = triangle; Sq =square; Tet = tetragon; Hex = hexagon; NR = nanorod; NW = nanowire; NT =nanotube; Nec = necklace-structure; C/S = core/shell structure; SF =snowflake-like particles; DD = dendrite; PP = porous particles; NH =nanohorn; Hol = hollow structure.

Preferred forms for platinum nanoparticles include cubic, cuboctahedron,dendrite and spherical particles.

Typically, when the ROS-quenching agent is a transition metal or atransition metal compound it present at a loading of 0.01 to 5 wt % ofthe membrane, preferably 0.1 to 5 wt %, more preferably 0.5 to 3 wt %.The loading can be calculated from the density of the ROS-quenchingagent, the uncoated/unimpregnated membrane, and the coated/impregnatedmembrane.

In some embodiments, the ROS quenching agent is present on the surfaceof the membrane. In alternative embodiments, the ROS quenching agent ispresent within the pores of the membrane.

ROS-Quenching Activity

In some embodiments the membrane used in the ROS-quenching barrier layerhas an ROS-quenching activity sufficient to quench a solution of H₂O₂having a concentration of 10 ppm, i.e. it can quench a solution having aH₂O₂ concentration of at least 10 ppm. The membrane typically has anROS-quenching activity sufficient to quench a solution of H₂O₂ having aconcentration of 20 ppm, preferably 50 ppm and more preferably 70 ppm,80 ppm or 100 ppm.

H₂O₂ concentrations are typically measured to an accuracy of +/−2 to 5ppm. Accordingly, as used herein, where an H₂O₂ concentration isindicated this is assumed to be stated to an accuracy of +/−5 ppm. A 400ppm H₂O₂ solution is prepared by diluting a 30% hydrogen peroxidesolution (133 μl) in UHP water (100 ml). This 400 ppm solution isdiluted to obtain the required concentration. Thus, a 10 ppm solution isobtained by diluting 1 ml of the 400 ppm solution in 39 ml of UHP water.Similarly, a 100 ppm solution is obtained by diluting 10 ml of the 400ppm solution in 30 ml UHP water.

ROS-quenching activity can be determined by passing up to 100 μl (e.g.100 μl) of a solution of H₂O₂ having a known concentration (e.g. 10 ppm,prepared as set out above) through a hollow fibre membrane (fibre: innerdiameter approx 410 μm; outer diameter approx 500 μm, typical length 25mm), e.g. using a needle, as schematically represented in FIG. 4, andmeasuring the H₂O₂ concentration of the solution once passed through themembrane. The solution is said to be quenched if the concentration ofH₂O₂ in the solution once passed through the membrane is less than about0.5 ppm. When measuring H₂O₂ concentration using standard reagent stripsknown in the art, the detection limit is approximately 0.5 ppm, so thatif detection is carried out using such a standard reagent strip, aquenched solution will have a concentration of H₂O₂ which isundetectable.

Oxidation of Analyte

The ROS-quenching membrane is typically capable of catalysing thedecomposition of ROS without substantially oxidising (or otherwisedepleting) analyte (e.g glucose). Thus, preferred ROS quenching agentsselectively oxidise ROS, such as H₂O₂, over analyte. In some embodimentstherefore, the membrane selectively quenches ROS. As used herein, amembrane which selectively quenches ROS typically has an ROS quenchingactivity sufficient to quench a solution having an H₂O₂ concentration ofat least 10 ppm, and substantially does not deplete analyte (e.g. it hasan analyte depletion rate of 1 mmol/hour or less, preferably 0.05mmol/hour or less and/or it depletes analyte by no more than 80%,preferably no more than 95% when analyte is passed through themembrane).

The extent of analyte depletion can be determined by passing a solutionof analyte (e.g. 100 to 500 μl) having a known concentration through awall of a membrane (fibre: inner diameter approx 410 μm; outer diameterapprox 500 μm, 0.5 mm, typical length 25 mm), e.g. using a needle, asschematically represented in FIG. 4, and measuring the analyteconcentration of the solution once passed through the membrane. Amembrane which substantially does not deplete analyte will typicallyproduce a concentration of analyte in the solution once passed throughthe membrane of 80% or more of that of the original solution, typically85% or more, preferably 90% or more, more preferably 95% or more, mostpreferably 99% or more.

Alternatively, the rate of depletion can be determined by placing themembrane in a solution of analyte having a known concentration anddetermining the analyte concentration at regular intervals. Typically,the analyte concentration is determined regularly (e.g. every 8 hours)over a period of at least 24 hours, preferably at least 48 hours. Atypical procedure is set out in Example 3, for determining glucosedepletion. A membrane which substantially does not cause depletion ofanalyte typically provides a rate of depletion of no more than 0.1 mmolanalyte per hour, preferably no more than 0.08 mmol/hour, morepreferably no more than 0.05 mmol/hour.

In some embodiments the extent or rate of analyte depletion iscontrolled by selecting an appropriate ROS-quenching agent, as describedabove.

In another embodiment the extent or rate of analyte depletion iscontrolled by selecting an appropriate ROS-quenching activity, asdescribed above.

In another embodiment the extent or rate of analyte depletion iscontrolled by selecting an appropriate method for producing theROS-quenching agent, as described below.

In another embodiment, the extent or rate of analyte depletion iscontrolled by selecting an appropriate form of nanoparticle, and/or byselecting an appropriate nanoparticle size, as described above.

The Sensor

An example of a sensor incorporating an optical fibre is depicted inFIGS. 1 and 1 a. The sensor 1 comprises an optical fibre 2 including asensing region 3 at its distal end. In the case of an invasive sensor,fibre 2 is adapted for insertion into a patient, for example insertioninto a blood vessel through a cannula. The sensing region 3 (depicted inmore detail in FIGS. 2 and 3) contains a cell or chamber 7 in which theindicator chemistry is contained. The optical fibre extends throughcable 4 to connector 5 which is adapted to mate with an appropriatemonitor 8. The monitor typically includes further optical cable 4 a thatmates with the connector at 5 a and at the other end bifurcates toconnect to (a) an appropriate source of incident light for the opticalsensor 9 and (b) a detector for the return signal 10.

In some embodiments, the sensor is a disposable sensor. The sensor istypically adapted to be connected to a non-disposable monitor includinga light source 9 and detector 10.

As depicted in FIG. 2, the sensing region 3 incorporates a cell 7 in theform of a chamber within the fibre. The cell may take any form, as longas it enables the indicator chemistry to be contained in the path of theincident light directed by the waveguide, here a fibre. Thus, the cellmay be attached to the distal end of the fibre or waveguide or may be inthe form of a chamber within the fibre having any desired shape.

The cell 7 contains the indicator chemistry. In the case of a glucosesensor, this is typically a boronic acid receptor for binding glucoseand a fluorophore associated with the receptor. The emission pattern(e.g. the wavelength, intensity, lifetime) of the fluorophore is alteredwhen the analyte is bound to the receptor allowing optical detection ofglucose. The description of the sensor will be given in detail hereinwith regard to a glucose sensor. However, it is to be appreciated thatthe ROS-quenching, analyte-permeable barrier layer can be applied tosensors other than glucose sensors.

Sensors having design features in addition to or different from thoseshown in the attached Figures are of course possible, provided thatthese include both of the required sensing region and barrier layer. Forexample, sensors such as those described and illustrated inWO2008/141241, WO2008/098087 and WO2011/113020 can be used.

The receptor and fluorophore may be directly bonded to one another as areceptor-fluorophore construct. Examples of suitable fluorophores aredescribed in WO 2010/116142, the content of which is incorporated hereinby reference, and include anthracene, pyrene and derivatives thereof.Examples of suitable boronic acid receptors are compounds having atleast one, preferably two boronic acid groups.

In a preferred embodiment, the receptor is a group of formula (I)

wherein m and n are the same or different and are typically one or two,preferably one; Sp is an alphatic spacer, typically an alkylene moiety,for example a C1-C12 alkylene moiety, e.g. a C6 alkylene moiety; and L1and L2 represent possible points of attachment to other moieties, forexample to a fluorophore or to a hydrogel. For example, L1 and L2 mayrepresent an alkylene, alkylene-arylene or alkylene-arylene-alkylenemoiety, linked to a functional group. Where no attachment to anothermoiety is envisaged, the functional group is protected or replaced by ahydrogen atom. Typical alkylene groups for L1 and L2 are C1-C4 alkylenegroups, e.g. methylene and ethylene. Typical arylene groups arephenylene groups. The functional group is typically any group which canreact to form a bond with, for example, the fluorophore or hydrogel,e.g. ester, amide, aldehyde or azide.

Varying the length of the spacer Sp alters the selectivity of thereceptor. Typically, a C6-alkylene chain provides a receptor which hasgood selectivity for glucose.

Further details of such receptors are found in U.S. Pat. No. 6,387,672,the contents of which are incorporated herein by reference.

Further examples of receptors suitable for the sensor include those offormula (II):

wherein X represents O, S, NR₂ or CHR₃;n is from 1 to 4;m is from 1 to 4, and n+m is 5;R₂ represents hydrogen or C₁₋₄ alkyl;each R₁ is the same or different and represents hydrogen, C₁₋₄ alkyl orC₃₋₇ cycloalkyl;or R₁, together with an adjacent R₁, R₂ or R₃ group and the carbon ornitrogen atoms to which they are attached, form a C₃₋₇ cycloalkyl or a5- or 6-membered heterocyclyl group,wherein when X represents CHR₃, R₃ together with an adjacent R₁ groupand the carbon atoms to which they are attached form a C₃₋₇ cycloalkylgroup. Further details of receptors of this type are found in U.S.61/431,756, the contents of which are incorporated herein by reference.

As used herein the term alkyl or alkylene is a linear or branched alkylgroup or moiety. An alkylene moiety may, for example, be one in whichfrom 1 to 15 carbon atoms are present such as a C₁₋₁₂ alkylene moiety,C₁₋₆ alkylene moiety or a C₁₋₄ alkylene moiety, e.g. methylene,ethylene, n-propylene, i-propylene, n-butylene, i-butylene andt-butylene. C₁₋₄ alkyl is typically methyl, ethyl, n-propyl, i-propyl,n-butyl or t-butyl. For the avoidance of doubt, where two alkyl groupsor alkylene moieties are present, the alkyl groups or alkylene moietiesmay be the same or different.

An alkyl group or alkylene moiety may be unsubstituted or substituted,for example it may carry one, two or three substituents selected fromhalogen, hydroxyl, amine, (C₁₋₄ alkyl) amine, di(C₁₋₄ alkyl) amine andC₁₋₄ alkoxy. Preferably an alkyl group or alkylene moiety isunsubstituted.

As used herein an arylene group is an unsaturated group which may bemonocyclic, bicyclic, or in which three or four fused rings may bepresent. Typically, an arylene group is phenylene. Arylene groups may beunsubstituted or substituted. Suitable substituents are C₁₋₄ alkylgroups, for example methyl and ethyl. Preferably, an arylene group isunsubstituted.

As used herein a C₃₋₇ cycloalkyl group is typically a cyclopentyl orcyclohexyl group. C₃₋₇ cycloalkyl groups may be unsubstituted orsubstituted. Suitable substituents are C₁₋₄ alkyl groups, for examplemethyl and ethyl. Preferably, a C₃₋₇ cycloalkyl group is unsubstituted.

As used herein a 5- or 6-membered heterocyclyl group is a 5- or6-membered saturated ring in which one or more, typically one or two,e.g. one, heteroatom selected from N, O and S is present. Preferredheterocyclyl groups are those in which a nitrogen atom is present, forexample piperidinyl and pyrrolidinyl. Heterocyclyl groups may beunsubstituted or substituted. Suitable substituents are C₁₋₄ alkylgroups, for example methyl and ethyl. Preferably, a heterocyclyl groupis unsubstituted.

The receptor and fluorophore are typically bound to one another and mayfurther be bound to a polymeric matrix such as a hydrogel, or to adendrimer. Examples of suitable hydrogels and dendrimers are thosedescribed in PCT/GB2011/000207, the content of which is incorporatedherein by reference.

Alternatively, the first receptor and first fluorophore may be notdirectly bonded to one another (for example, they may be not bonded toone another or they may be bonded only via a polymeric chain such as apolymeric chain contained within a hydrogel matrix). It will be clearthat when the first receptor and first fluorophore are not directlybonded to one another, they must still be capable of interacting in sucha way that the fluorescence behaviour of the first fluorophore changeswhen the indicator system is exposed to glucose. For example, the firstfluorophore and the second fluorophore may be capable of bindingelectrostatically (e.g., as a charge pair), which binding is capable ofbeing at least partly disrupted by the presence of glucose. Examples ofsuitable first fluorophores include pyranine (HPTS) and its derivatives,such as HPTS itself and the derivatives HPTS-PEG, HPTS-MA, HPTS-CO₂,HPTS-TriCys-MA and HPTS-LysMA disclosed in US 2009/0177143, the contentof which is herein incorporated by reference in its entirety. Furthersuitable first fluorophores may include the SNAF and SNAFL dyescommercially available from Molecular Probes. Examples of suitable firstreceptors include aromatic boronic acids covalently bonded to aconjugated nitrogen-containing heterocyclic aromatic bis-onium structure(e.g. a viologen). Examples of such first receptors are provided in US2009/0177143, the content of which is herein incorporated by referencein its entirety. One particularly suitable first receptor is 3,3′-oBBV,as described in US 2009/0177143.

The sensing region 3 of the glucose sensor has one or more openings 6 a,6 b to enable glucose to enter the cell. The barrier layer can beprovided across these openings so that the sample under test enters thecell through the barrier layer. In FIGS. 2 and 3, the barrier layer isprovided over the entire sensing region 3. Alternatively, however, thebarrier layer may be provided on only part of the sensing region, forexample only across openings 6 a and 6 b.

The sensor is typically designed such that any openings into the sensingregion through which the sample under test can pass are covered with thebarrier layer. This ensures that passage of H₂O₂ into the sensing regionis restricted or prevented. In some embodiments, the entire sensingregion, or the entire surface of the sensor which is to come intocontact with the sample under test, is coated or sheathed with thebarrier layer.

As depicted in FIG. 2, the barrier layer BL may be applied directly ontothe sensing region, here onto the tip of the optical fibre. Thisembodiment is appropriate, for example, where the barrier layer is adialysis membrane. In an alternative embodiment depicted in FIG. 3, thesensing region 3 is provided on a separate support 11. The separatesupport structure can provide additional strength compared with theapplication of the barrier layer directly to the sensing region, andthis embodiment is therefore also appropriate for use with dialysismembrane barrier layers. Holes or pores are provided in the support toenable glucose to enter the sensing region 3. Suitable supportstructures are polymer tubes which are perforated with holes, forexample by laser ablation. Microporous hollow fibres which are commonlyused in medical oxygenators and which have pores of approximately 0.2micron in diameter provide appropriate support structures for use withfibre optic sensors. Alternative support structures are woven sheaths ofpolymeric or metallic materials such as those described inWO2009/019470, the contents of which are incorporated herein byreference in their entirety.

In some embodiments, as depicted in FIG. 3 a, the barrier layer itselfmay form the support structure (BL/11). Preferably, in this embodiment,the membrane used to form the barrier layer is a microporous hollowfibre membrane.

If desired, the barrier layer may be adhered to the surface of thesensor e.g. to the optical fibre itself, or, where relevant, to theseparate support structure. This can be achieved by application of asuitable adherent such as cyanoacrylate. Alternatively, where the sensorsurface and the barrier layer material are appropriate, the jointbetween the barrier layer and the sensor can be thermoformed, e.g. atJa, Jb of FIGS. 2 and 3.

Method of Manufacture

The sensor is manufactured by providing a sensing region includingsuitable indicating chemistry (e.g. in the case of a glucose sensor aboronic acid receptor for binding to glucose and a fluorophoreassociated with said receptor); and providing an ROS-quenchinganalyte-permeable barrier layer on at least a part of the sensingregion; and wherein the sensor is adapted so that analyte enters thesensing region of the sensor through said barrier layer. In the case ofan optical sensor the method of manufacture also includes providing anoptical waveguide fro directing incident light onto the sensor.

In some embodiments, the membrane used in the ROS-quenching analytepermeable barrier layer is formed by vapour deposition. In thisembodiment, the ROS-quenching agent is typically a metal or an alloy andthe metal or alloy is sputtered under vacuum and at low temperature toform a metal vapour which can be directed toward the membrane fordeposition on the membrane surfaces, including those within the pores ofthe membrane structure.

In an alternative embodiment, the membrane used in the ROS-quenchinganalyte permeable barrier layer is formed by a method including (i)coating or impregnating a semi-permeable membrane, as described above,with an ROS-quenching agent, (ii) washing the membrane and (iii) dryingthe membrane.

In some embodiments, step (i) comprises wetting a membrane in a suitablewater miscible solvent followed by shaking. In a preferred example ofthis embodiment, the solvent is capable of solvating the pores of themembrane. In some examples of this embodiment the solvent is a polarnon-protic solvent. In some examples of this embodiment the solvent ishydrophobic but water soluble. Specific examples of solvents capable ofsolvating the pores of the membrane include N-methylpyrrolidone (NMP),dimethylsulfoxide (DMSO) and dimethylformamide (DMF). Wetting a membranewith a solvent capable of solvating the pores of the membrane isparticularly effective in enabling the ROS quenching agent to bedistributed along the length of the pores.

In some embodiments, step (i) comprises immersing a membrane in asolution containing a species capable of forming an ROS-quenching agent(an ROS-quenching precursor) and subsequently subjecting the membrane toconditions such that the H₂O₂-quenching agent forms on or in themembrane. In a preferred example of this embodiment, when theH₂O₂-quenching agent is platinum, step (i) comprises soaking themembrane in a solution of a platinum containing salt, such as a tetra-or hexa-chloroplatinate salt e.g. sodium or potassiumtetrachloroplatinate or sodium or potassium hexachloroplatinate,followed by reduction of the platinum containing salt to platinum metalwith a reducing agent such as formic acid, ascorbic acid or hydrazine,preferably formic acid or ascorbic acid, more preferably formic acid.When formic acid is used as the reducing agent, the ROS-quenchingprecursor is reduced to form an ROS-quenching agent, and the formic acidis oxidised to carbon dioxide gas. This avoids any residue from thereducing agent remaining on the ROS-quenching agent after formation.

In some embodiments the reducing agent may be ascorbic acid orhydrazine, preferably ascorbic acid.

In some embodiments, step (i) comprises shaking and/or heating themembrane whilst under reducing conditions. Thus, for example, themembrane may be contacted with the reducing agent and shaken for aperiod of up to 7 days, for example at least 2 hours, at least 12 hoursor at least 24 hours. Shaking is, for example, carried out at 200 rpm ormore, for example up to 400 rpm. This can be achieved at an amplitude of25 mm on an orbital incubator.

Heating may be carried out concurrently with shaking. Alternatively,either heating or shaking alone is used. Where the membrane is heatedwhilst under reducing conditions, typically heating is at a temperatureof up to 45° C. for a period of up to 7 days, for example at least 2hours, at least 12 hours or at least 24 hours.

Step (i) may further comprise a washing step (separate from the washingstep (ii)). Step (i) may be repeated one or more times, e.g. 1, 2, or 3times. Where the membrane is immersed in a solution, sonication may beapplied to ensure full wetting of the membrane.

Step (i) may further comprise a second reduction, typically after awashing step as described above. Suitable reducing agents for the secondreduction include hydrazine and salts of Group 13 hydrides salts such asborohydride salts and aluminium hydride salts, sodium borohydride andlithium aluminiumhydride, preferably sodium borohydride. Preferably thesecond reducing reagent is hydrazine.

Step (ii) typically comprises soaking the coated or impregnated membranein water. Typically, the membrane is soaked for at least 12 hours, e.g.at least 24 hours or at least 36 hours. Typically, the water is at atemperature of from 27-47° C., preferably 32-42° C., more preferably36-38° C. and most preferably about 37° C. Step (ii) typically furthercomprises immersing the membrane in an organic solvent one or moretimes, e.g 1, 2, 3, 4 or more times. Preferably the organic solvent isan aliphatic C1 to C6 alcohol, more preferably ethanol.

In some embodiments, step (iii) is carried out under reduced pressure,preferably under vacuum, for one hour or more. In an alternativeembodiment, step (iii) is carried out at elevated temperature,preferably at 40-50° C., e.g. about 45° C., for two hours or more.

In some embodiments, the method comprises contacting a barrier layerwith a ROS-quenching precursor and a reducing agent; and reducing theROS-quenching precursor to form a ROS-quenching agent on or in thebarrier layer. The process may optionally be repeated one or more times(e.g 1, 2, or 3 times) to provide further layers of the ROS-quenchingagent.

In some embodiments the reducing agent is formic acid.

An example of this embodiment is as follows:

A polypropylene hollow fibre membrane is fully wetted by adding asuitable water miscible solvent (e.g. N-methylpyrrolidone, NMP) followedby shaking. The solvent is removed from the membrane and fresh solvent(e.g. NMP) is added to the membrane followed by shaking. Theremoval/replacement/shaking process may be repeated, e.g. three or moretimes.

The solvent is removed and the following are added to the membrane, withshaking between each addition:

-   -   a suitable water miscible solvent    -   UHP water    -   0.05-0.50 mmol (e.g 0.1 mmol) of a platinum containing salt        (e.g. a tetra- or hexa-chloroplatinate salt e.g. sodium or        potassium tetrachloroplatinate or sodium or potassium        hexachloroplatinate)    -   formic acid

The membrane, in the solution, is shaken at elevated temperature (e.g 30to 60° C., typically 40-50° C.) for 12 to 24 hours

The membrane is washed repeatedly (e.g a minimum of 5 times) in anappropriate solvent system (e.g a mixture of water and a water miscibleorganic solvent, typically IPA).

The membrane is dried in air at ambient temperature for at least onehour.

In some embodiments, the method comprises contacting a barrier layerwith a ROS-quenching precursor and a first reducing agent; partiallyreducing the ROS-quenching precursor to form a ROS-quenching agent on orin the barrier layer; contacting the barrier layer with a secondreducing agent; fully reducing the remaining ROS-quenching precursor toform a ROS-quenching agent on or in the barrier layer. The process mayoptionally be repeated one or more times (e.g 1, 2, or 3 times) toprovide further layers of the ROS-quenching agent.

In some embodiments the first reducing agent is ascorbic acid and thesecond reducing agent is hydrazine. In another embodiment, the firstreducing agent is ascorbic acid and the second reducing agent is aborohydride salt, e.g. sodium borohydride.

An example of this embodiment is as follows:

A polypropylene hollow fibre membrane is fully wetted by adding asuitable water miscible solvent (e.g. propanol). The solvent is removedfrom the membrane and water is added. This step is repeated until themembrane is fully wetted in water.

0.05-0.10 mmol (e.g 0.06 mmol) of a platinum containing salt (e.g. atetra- or hexa-chloroplatinate salt e.g. sodium or potassiumtetrachloroplatinate or sodium or potassium hexachloroplatinate),ascorbic acid, and a concentrated inorganic acid (e.g. HCl) is added tothe solution containing the membrane. The membrane, in the solution, iskept at elevated temperature (e.g 30 to 60° C., typically 40-50° C.) forat least 4 days (e.g 4-7 days, typically about 6 days).

The membrane is washed repeatedly (e.g a minimum of 5 times) in anappropriate solvent system (e.g a mixture of water and a water miscibleorganic solvent, typically IPA). NaBH₄ is added to the membrane in in anappropriate solvent system (e.g a mixture of water and a water miscibleorganic solvent, typically IPA).

The above steps are repeated, except that the membrane, in thePt-containing salt/ascorbic acid/inorganic acid solution, is kept atelevated temperature (e.g 30 to 60° C., typically 40-50° C.) for about 1day.

The membrane is dried in air at ambient temperature for at least onehour.

Another example of this embodiment follows the procedure set out aboveexcept that for the first addition of the Pt-containing salt, 0.10-0.20mmol (e.g 0.12 mmol) is used; and a mixture of a concentrated inorganicacid (e.g. HCl) and hydrazine is used instead of NaBH₄.

Use of the Sensor

The present sensor may be used by inserting the sensor into a sample,for instance a sample of body fluid or tissue, e.g. blood, providingincident light to the sensing region of the sensor and detecting theemission pattern of the fluorophore from the emission pattern of thefluorophore. As described above, the emission pattern (e.g. thewavelength, intensity, lifetime) of the fluorophore is altered whenglucose is bound to the receptor, allowing an amount of glucose in thesample to be detected and/or quantified.

EXAMPLES

ROS-quenching membranes were produced according to the methods set outbelow.

Example 1 First Application Wetting

A polypropylene hollow fibre membrane (fibre internal diameter 416micron, outer diameter 510 micron, length 25 mm) was fully wetted byadding propanol (3 ml). The solvent was removed from the membrane andUHP water (3 ml) was added. This step was repeated at least 5 timesuntil the membrane was fully wetted in UHP water (3 ml).

First Application—First Reducing Agent

The UHP water was removed from the vial containing the membrane andfresh UHP water (2 ml) added followed by potassium tetrachloroplatinate(50 mg), ascorbic acid (300 mg), and 37% HCl (0.25 ml).

First Application—First Reduction

The membrane, in the Pt/ascorbic acid solution, was placed in an ovenand heated at 45° C. for 6 days.

First Application—Second Reducing Agent and Second Reduction

The membrane was washed a minimum of 5 times in an IPA/UHP watersolution (70:30 v/v, 7 ml). NaBH₄ (50 mg) was added to the membrane inan IPA/UHP water solution (70:30 v/v, 7 ml).

First Application—Washing

The membrane was washed a minimum of 5 times in an IPA/UHP watersolution (70:30 v/v, 7 ml). The membrane was fully wetted by addingpropanol (3 ml). The solvent was removed from the membrane and UHP water(3 ml) was added. This step was repeated at least 5 times until therewas no propanol present.

Second Application—First Reducing Agent

The UHP water was removed from the vial containing the membrane andfresh UHP water (2 ml) added followed by potassium tetrachloroplatinate(25 mg), ascorbic acid (300 mg), and 37% HCl (0.25 ml).

Second Application—First Reduction

The membrane, in the Pt/ascorbic acid solution, was placed in an ovenand heated at 45° C. for 1 day.

Second Application—Second Reducing Agent and Second Reduction

The membrane was washed a minimum of 5 times in an IPA/UHP watersolution (70:30 v/v, 7 ml). NaBH₄ (50 mg) was added to the solutioncontaining the membrane.

Second Application—Washing

The membrane was washed a minimum of 5 times in an IPA/UHP watersolution (70:30 v/v, 7 ml). After water washing the membrane was dippedin ethanol (7 ml).

Drying

The membrane was dried under air at room temperature and pressure for aminimum of 1 hour.

Example 2

An ROS quenching membrane was produced following the procedure ofExample 1 except that:

-   -   (v) for the First application—First reducing agent step 50 mg of        potassium tetrachloroplatinate was used; and    -   (vi) for the First application—Second reducing agent        steps—Second reducing agent and second reduction steps, a        mixture of 37% HCl (0.5 ml) and hydrazine (1.5 ml) was used        instead of NaBH₄ in an IPA/UHP water solution (70:30 v/v, 7 ml).

SEM images of the membrane are shown in FIG. 5. A and B are surfaceimages whilst C and D are cross-sectional images.

Example 3

Hydrogen peroxide solution (10 ppm) was prepared as follows:

A 30% hydrogen peroxide solution (133 μl) was diluted in UHP water (100ml) to obtain a 400 ppm solution of hydrogen peroxide. To obtain a 10ppm solution of hydrogen peroxide 1 ml of the 400 ppm solution wasdiluted in 39 ml of UHP water. 100 μl of the 10 ppm solution thusprepared was pumped through the wall of a 25 mm length of the membraneof Example 1, as schematically depicted in FIG. 4. No peroxide wasdetected in the solution that had travelled through the membrane.Detection of H₂O₂ was carried out using peroxide test strips capable ofdetecting 0.5 ppm or greater peroxide (e.g. EM Quant™). The sameprocedure was repeated with the membranes of Example 2 and the peroxidelevels in the solution which had travelled through the membrane wasbelow detection levels (0.5 ppm).

Glucose depletion of the membrane is tested as follows:

Prepare a D-glucose solution (5 mM) and allow the anomeric ratio toequilibrate (40:60 α/β ratio) before use. Place 10 lengths of themembrane to be tested in a 3.5 ml vial and add the pre-preparedD-glucose solution (3 ml). Add the pre-prepared D-glucose solution (3ml) to a second, empty, 3.5 ml vial as a control. Measure the D-glucoseconcentration in both vials using a YSI 2300 Stat plus then incubate thesamples at 37° C. Over a minimum period of 24 hours measure theD-glucose concentration a minimum of 3 times. Plot a graph of time vsD-glucose concentration and determine the rate of D-glucose degradation.

Example 4

20 lengths (25 mm each length) of the membrane prepared according to theprocess of Example 2 were placed in a 4.2 mM solution of glucose (3 ml).Glucose concentration was measured against time. After 72 hours, theglucose concentration was increased to 7.4 mM. The results of glucoseconcentration measurements are shown in FIG. 7. The vertical arrowsindicate were the glucose concentration was increased.

The results show no drop in glucose concentration, showing that glucoseis stable in the presence of the membranes.

Example 5

ROS-quenching membranes were produced by the processes of Examples 1 and2 using either a single application of Pt or two applications of Pt. Formembranes having a single application of Pt the Second application stepswere omitted. The ROS-quenching activities of the membranes weredetermined by measuring the rate of evolution of oxygen when a 2.5 cmlength of the membrane was placed into a 30% hydrogen peroxide solution.The results are given in Table 1 below. Platinum loading is given forcertain membranes.

TABLE 1 Pt loading/ O₂ evolved/ Membrane wt % cm³ · min⁻¹ Process Notes1 0.5  12.3 Ex 1 Single application 2 1.67 19.4 Ex 1 Two applications 3— 26.0 Ex 2 Two applications

Example 6 In Vitro Testing

An optical glucose sensor having an indicator system including adi-boronic acid and a fluorophore associated therewith was constructedwith a platinised membrane prepared in accordance with Example 2, suchthat analyte entering the sensor passed through the membrane. Thesensing portion of the sensor was inserted into a 10 ppm hydrogenperoxide solution prepared in accordance with the procedure described inExample 3. The glucose concentration was monitored on a continuous basisusing the sensor and also monitored every 5 minutes using anelectrochemical glucose sensor (YSI 2300 stat). Testing was continuedfor 60 minutes and the results are shown in FIG. 6 (solid line).

A corresponding experiment was carried out using an identical sensor,with the exception that the membrane used was not platinised. Theresults are also depicted in FIG. 6 (dotted line).

Example 7 Effect of Exposure to Peroxide

Platinised and non-platinised sensors as described in Example 6 werecalibrated for glucose both before and after exposure to 10 ppm hydrogenperoxide solution. The results are depicted in Table 2 below. Nosignificant changes were observed between the two sensor calibrations ofthe platinised sensor. In contrast, significant degradation was seen inthe non-platinised sensor.

TABLE 2 Calibration constants before and after exposure to peroxide. I₀and I_(∞) have been normalised. Steril- Batch ised I₀ I_(∞) K Mod_(5 mM)% Platinised Yes Before 1.000 3.679 0.036 28.9 membrane After 1.0003.712 0.035 28.7 sensors +/−% −0.3 0.6 −2.2 −0.5 Non- Yes Before 1.0002.858 0.033 20.9 platinised After 1.000 1.612 0.024 6.2 membrane +/−%−22.7 −56.4 −26.2 −70.1 sensors

Example 8 In Vivo Testing

An optical glucose sensor having an indicator system including adi-boronic acid and a fluorophore associated therewith was constructedwith a platinised membrane prepared in accordance with Example 2, suchthat analyte entering the sensor passed through the membrane. Thesensing portion of the sensor was inserted into the vein of a patientvia a 18G cannular. The glucose concentration as determined by thesensor was recorded on a continuous basis. Whilst testing was carriedout, blood samples were taken from the patient approximately every 2hours, or as needed, and the glucose concentration of each sampledetermined using an electrochemical glucose sensor (YSI 2300 stat). Theresults are shown in FIG. 9 b.

A corresponding experiment was carried out using an identical sensor,with the exception that the membrane used was not platinised. Theresults are shown in FIG. 9 a. As is apparent from the figures, theresults from the sensor having a platinised membrane correspond wellwith those from the YSI stat, whereas the non-platinised sensor does notshow close correspondence with the YSI stat results over the testperiod.

Example 9

Three sensors were constructed having membranes produced according tothe following process:

-   1. A 25 mm length of polypropylene hollow fibre membrane (MPHF) was    placed into a 7 ml vial.-   2. The MPHF membrane was fully wetted by adding NMP (2 ml) and the    vial shaken. This wetting process was instantaneous and the membrane    become translucent on wetting.-   3. The solvent in the vial was removed and immediately replaced with    NMP (2 ml). The vial was shaken to wash the membrane. This    removal/replacement and shaking process was repeated a minimum of    three times.-   4. The NMP was removed from the vial and the following were added    with shaking between each addition:    -   NMP (2 ml)    -   UHP Water (4 ml)    -   Potassium (II) tetrachloroplatinate (0.75 ml of a 50 mg.ml-1        solution)    -   Formic acid (0.1 ml)-   5. The vial was placed in a heated shaker at 45° C. for a minimum of    12 hours and a maximum of 24 hours.-   6. The solvent in the vial was removed and replaced with 70:30    IPA/UHP water (7 ml). This process was repeated a minimum of 5 times    until the washings were clear.-   7. The membranes were removed from the vial and dried for a minimum    of one hour at ambient conditions supported on a straight wire to    keep the membranes straight.

The sensors were exposed to a 10 ppm solution of hydrogen peroxide for 1hour, as was an unplatinised control sensor, and the glucoseconcentration measured. (FIG. 10 shows results for the platinisedsensors 1, 2 and 3 as IB070-001, IB070-002, and IB070-003 respectively.Results for the unplatinised control are shown as IB066-011.

These sensors were calibrated before and after this test. Table 3 is acomparison of these calibrations.

TABLE 3 Percentage changes in calibration constants for platinisedsensors containing membrane produced according to the procedure ofExample 9 (sensors 1-3) when exposed to a 10 ppm hydrogen peroxidesolution, as compared to an unplatinised sensor (control) Sensor 1 2 3Control I₀ −0.4 −0.8 −1.1 −21.8 I_(∞) 2.7 0.9 0.2 −49.0 K −5.2 −3.0 −0.5−16.4 Mod_(5 mM) −0.2 −0.1 1.0 −48.1

The platinised sensors appeared to be fully resistant to the hydrogenperoxide solution and there was no significant change in theircalibration constants before and after the test. The average modulationat 5 mM glucose changed from 27.47% to 27.53%.

The ROS quenching ability of membranes produced according to the aboveprocess was determined in 10 ppm H₂O₂ using the procedure of Example 3.The peroxide levels in the solution which had travelled through themembrane was below detection levels (0.5 ppm).

The present invention has been described with reference to a number ofparticular embodiments and examples. The invention is not, however,limited to these specific embodiments and examples.

The invention claimed is:
 1. A process for producing a Reactive OxygenSpecies (ROS)-quenching analyte-permeable membrane suitable for use inan optical sensor for detecting and/or quantifying the amount of analytein a sample comprising: a. contacting a polypropylene hollow fibremembrane with a ROS-quenching precursor and a reducing agent; b.reducing the ROS-quenching precursor within pores of the polypropylenehollow fibre membrane to form a ROS-quenching agent within the pores ofthe polypropylene hollow fibre membrane; and optionally repeating steps(a) and (b).
 2. The process according to claim 1 further comprising,before step (a): (i) contacting the polypropylene hollow fibre membranewith a ROS-quenching precursor and a preliminary reducing agent; (ii)partially reducing the ROS-quenching precursor within the pores of thepolypropylene hollow fibre membrane to form a ROS-quenching agent withinthe pores of the polypropylene hollow fibre membrane; and optionallyrepeating steps (i), (ii), (a) and (b).
 3. The process according toclaim 2 wherein the preliminary reducing agent is ascorbic acid.
 4. Theprocess according to claim 2 wherein the reducing agent is hydrazine. 5.The process according to claim 1 wherein the (ROS)-quenchinganalyte-permeable membrane is suitable for use in an optical sensor fordetecting or quantifying an amount of glucose in a sample.
 6. Theprocess according to claim 1 wherein the ROS-quenching precursor is asalt of a Group 10 or 11 metal-containing ion and the ROS-quenchingagent is a Group 10 or 11 metal.
 7. The process according to claim 6wherein the Group 10 or 11 metal is platinum.
 8. The process accordingto claim 7 wherein the ROS-quenching precursor is a tetrachloroplatinatesalt.
 9. The process according to claim 7 wherein the platinum is in theform of nanoparticles.
 10. The process according to claim 1 wherein thereducing agent is formic acid.
 11. The process according to claim 1,wherein the ROS-quenching agent is distributed along the length of eachpore of the polypropylene hollow fibre membrane.
 12. The processaccording to claim 1, wherein the ROS-quenching agent is located tightlywithin the pores of the polypropylene hollow fibre membrane as a resultof the process of reducing the ROS-quenching precursor within the poresof the polypropylene hollow fibre membrane so that the quenching agentdoes not leach out from the pores during use of the membrane.
 13. Aprocess for producing a Reactive Oxygen Species (ROS)-quenchinganalyte-permeable membrane on a sensor for detecting and/or quantifyingthe amount of analyte in a sample comprising: (a) contacting apolypropylene hollow fibre membrane with a ROS-quenching precursor and areducing agent; (b) reducing the ROS-quenching precursor within pores ofthe polypropylene hollow fibre membrane to form a ROS-quenching agentwithin the pores of the membrane; and optionally repeating steps (a) and(b), and further comprising providing a sensor comprising a sensingregion, wherein the sensor is suitable for detecting and/or quantifyingthe amount of analyte in a sample; forming the ROS-quenchinganalyte-permeable membrane into a barrier layer on at least a part ofthe sensing region and wherein the sensor is adapted so that analyteenters the sensing region of the sensor through said barrier layer. 14.The process according to claim 13 wherein said sensor comprises: asensing region; and a barrier layer comprising a reactive oxygen species(ROS)-quenching, analyte-permeable membrane having an ROS-quenchingagent adsorbed thereto and wherein the membrane has a ROS-quenchingactivity sufficient to quench a solution of H₂O₂ having a concentrationof 10 ppm.
 15. A reactive oxygen species (ROS)-quenching,analyte-permeable membrane, suitable for use in an optical sensor fordetecting and/or quantifying the amount of analyte in a sample, themembrane having an ROS-quenching activity sufficient to quench asolution of H₂O₂ having a concentration of 10 ppm, wherein said membraneis obtainable by a process comprising: (a) contacting a polypropylenehollow fibre membrane with a ROS-quenching precursor and a reducingagent; (b) reducing the ROS-quenching precursor within pores of thepolypropylene hollow fibre membrane to form a ROS-quenching agent withinthe pores of the polypropylene hollow fibre membrane; and optionallyrepeating steps (a) and (b).
 16. The membrane according to claim 15,wherein the ROS-quenching agent is distributed along the length of eachpore of the polypropylene hollow fibre membrane.
 17. The membraneaccording to claim 15, wherein the ROS-quenching agent is locatedtightly within the pores of the polypropylene hollow fibre membrane as aresult of the process of reducing the ROS-quenching precursor within thepores of the polypropylene hollow fibre membrane so that the quenchingagent does not leach out from the pores during use of the membrane. 18.A sensor for detecting and/or quantifying the amount of analyte in asample, the sensor comprising: a sensing region; and a barrier layercomprising a reactive oxygen species (ROS)-quenching, analyte-permeablemembrane having an ROS-quenching agent adsorbed thereto; wherein thesensor is adapted so that the sample enters the sensing region of thesensor through said barrier layer, wherein the ROS-quenching,analyte-permeable membrane is obtainable by a process comprising: (a)contacting a polypropylene hollow fibre membrane with a ROS-quenchingprecursor and a reducing agent; (b) reducing the ROS-quenching precursorwithin pores of the polypropylene hollow fibre membrane to form aROS-quenching agent within the pores of the polypropylene hollow fibremembrane; and optionally repeating steps (a) and (b).
 19. The sensoraccording to claim 18, wherein the ROS-quenching agent is distributedalong the length of each pore of the polypropylene hollow fibremembrane.
 20. The sensor according to claim 18, wherein theROS-quenching agent is located tightly within the pores of thepolypropylene hollow fibre membrane as a result of the process ofreducing the ROS-quenching precursor within the pores of thepolypropylene hollow fibre membrane so that the quenching agent does notleach out from the pores during use of the sensor.